The determination of serum copper is of primary importance in a large number of diseases as for example in the Alzheimer's disease (AD). Alzheimer's disease is a neurological disorder characterized by memory loss and progressive dementia. The late onset form of the disease is sporadic and has a complex disease aetiology, with familiarity and age as the most widely accepted risk factors. The cause of the disease appears closely related to the aggregation within the brain of the beta-amyloid (Aβ) peptide and tau proteins in neurofibrillary tangles. Moreover, the epsilon 4 allele of the apolipoprotein E (APOE) gene has been proven to increase Alzheimer's Disease risk and decrease the age of onset, even though it accounts only for a percentage of Alzheimer's Disease heritability, leaving several genetic risk factors to be identified. The ‘amyloid cascade’, which has been claimed as the most popular Alzheimer hypothesis, has now taken many forms as new details about the disease emerge. In fact, diverse pathogenetic pathways have been postulated to contribute to Alzheimer's Disease onset and progression. For example, besides Aβ oligomers and toxic tau aggregates, oxidative damage, aberrant inflammation or impaired energy metabolism have been pointed out among the pathogenic pathways involved in the disease cascade that need to be targeted. There is abundant evidence that oxidative stress, mainly via metal redox reactions, can cause damage to the Alzheimer's Disease brain. Specifically, it has been proposed that the hyper metallization of the Aβ peptide can be at the basis of redox cycles of oxidative stress and H2O2 production, Aβ oligomer formation and precipitation. A derangement of metal homeostasis leading to a labile pool of copper may feed the brain copper reservoir which can enter Aβ-oxidative stress cycles, generating pleiotropic effects on the Alzheimer's Disease cascade. This notion is now sustained by diverse lines of evidence showing that copper is slightly but significantly increased in Alzheimer's Disease, that a specific and relative high increase in the serum pool of copper non-bound to ceruloplasmin (also named ‘free’ copper), is associated with some typical signs and cerebrospinal fluid markers of Alzheimer's Disease and it is already evident in subjects complaining very mild cognitive impairments. Even though copper dysfunction cannot be assumed as the only determinant of the disease, its causative, rather than associated, role as an important risk factor for Alzheimer's Disease can be claimed, since it appears sustained by solid clinical, epidemiological, experimental, meta-analysis and genetic evidence.
Ceruloplasmin is the major copper-carrying protein in the blood, it is the main ferroxidase protein in serum, and it binds structurally 6 atoms of copper, to form an active holo-form of the protein, which can account for 85-95% of circulating copper, the remaining being free copper. In fact in previous studies the inventors used to estimate free copper starting from copper and ceruloplasmin measures, with the calculation as follows: serum copper concentration were double check by measured them either with the atomic absorption spectroscopy technique utilizing a Perkin Elmer AANALYST 300 atomic absorption spectrophotometer equipped with a graphite furnace with platform HGA 800 graphite tube, or according to the colorimetric method of Abe et al. Clin Chem 1989 (Randox Laboratories, Crumlin, UK); ceruloplasmin was analyzed by immunoturbidimetry assay (Horiba ABX, Montpellier, France) according to Wolf PI Crit Rev Clin Lab Sci 1982, for each serum copper and ceruloplasmin pair it has been computed the amount of copper bound to ceruloplasmin (CB) and the amount of copper not bound to ceruloplasmin (‘free’ copper) following standard procedures disclosed in Walsh et al. Ann Clin Biochem 2003. This calculation expresses ‘free’ copper in μmol/L and is based on the evidence that ceruloplasmin contains 0.3% copper.
Moreover, the inventors have recently set up an automated procedure to measure ceruloplasmin oxidase activity which utilizes o-diansidine dihydrochloride as a substrate, according to previous methods. In fact, it is well known that values of ceruloplasmin obtained immunologically, as we showed them in our previous studies, result in higher values than those obtained enzymatically, i.e., monitoring the protein's oxidase activity. This is because the apo-form of ceruloplasmin is biologically inactive. As a consequence the values of free copper derived by the method and calculation reported above are underestimated.
However, quantification of ceruloplasmin by the enzymatic method based on standard ceruloplasmin solutions has not been considered in the state of the art, because of its cost, the variable purity of commercially available ceruloplasmin and the general recommendation to report serum enzymes in International Units (UI). Previously the inventors have tried to quantify the amount of ceruloplasmin starting from the protein's oxidase activity with a commercial standard (Human Serum Ceruloplasmin, Sigma-Aldrich), but the spectroscopic inspection of the latter revealed a decay in the protein peak of absorbance in day-to-day assays, decreasing the confidence in using the enzymatic detection to quantify the protein amount, necessary to estimate the free copper value.
Hyo Sung Jung et al. (J. Am. Chem. Soc. 2009) disclosed the synthesis and the use of different coumarin probes for the determination of free copper in biological systems.
Scope of the present invention is to provide novel methods and kits to measure directly free copper in serum avoiding the underestimation or overestimation of free copper that occur in the methods of prior art.